The overall objective of the proposed program is the mechanistic elucidation of the key enzymatic steps in the biosynthesis of carnitine in mammals. This objective has significant biochemical and physiological interest because any nutritional or pathological interference with the biosynthesis of camitine may lead to serious metabolic difficulties specifically with regard to lipids. In the framework of that objective we shall first concentrate our efforts on determining the mechanism of the last step in camitine biosynthesis, namely the hydroxylation of gamma- butyrobetaine to carnitine by what is known to be an a-ketoglutarate-dependent dioxygenase that requires the participation of molecular oxygen, ferrous ions and a reducing agent such as ascorbic acid. We have already developed an assay based on the detritiation of tritiated gamma-butyrobetaine. Using that assay, the purification of the hydroxylase from calf liver and a bacterial source will be monitored. Ultimately the purified enzyme will be studied for kinetics and mechanism of action. That hydroxylation occurs stereospecifically with retention of configuration, has already been established, however, whether a peroxy or other kind of derivative intervenes awaits further studies. By use of substrate amounts of enzyme we shall attempt to uncouple the decarboxylation of alpha-ketoglutarate from the hydroxylation of gamma-butyrobetaine. We shall try to determine why catalase and other proteins enhance the hydroxylation. The hydroxylation by a similar dioxygenase of 6-Trimethyl-L-lysine to 3-hydroxy-6-trimethyl-L-lysine, and the enzymology of its cleavage and subsequent oxidation leading to the formation of gamma-butyrobetaine will also be investigated. Finally, we shall determine whether any of these reactions proceed in cultured cells such as human lung fibroblasts, calf aorta and human uterine muscle cells and 3T3 L-1 preadipocyte cells. These cells will also be compared with respect to their capacity for uptake and concentration of carnitine.